The long term objective of this proposal is the understanding of voltage sensing in membrane proteins at the molecular level. Voltage sensing plays a major role in excitable tissues such as nerve, muscle and heart. We propose to study how voltage changes induce conformational changes in voltage gated sodium and potassium channels and in the voltage sensitive phosphatase Ci-VSP by correlating the gating currents with simultaneous rearrangements followed by fluorescence changes of probes placed in specific sites. Ultimately we would like to reproduce the function of the proteins with the landscape of energy obtained from structural changes. In this period we have three specific aims. Aim 1: Correlation of voltage sensor regions with the energy landscape of activation. Using Shaker K channels as a model we will study the trajectory of the gating charges through the hydrophobic plug using a fluorescent replacement of arginine that is quenched by Trp. Site-directed electrochromic fluorometry will be used to test the local field in different states of the sensor during gating. The interactions of the charges with the plug will be studied during activation and deactivation as a function of time and voltage. We will look for possible movements of S2 and S3, try to define the differences between the activated and relaxed states using LRET, and search for conditions that stabilize the relaxed state. Aim 2: Correlation of conformations and kinetics with Ci-VSP structures. Recent crystal structures of Ci-VSP in putative resting and activated/relaxed states will be compared and correlated with the function and structural changes detected during transitions to address how many charges move per molecule, the size of the individual shot of charge during gating, the effect of the residues of the hydrophobic plug on kinetics and steady-state, a possible secondary structure change of S4 during gating, and recording of single sensor movements by single molecule fluorescence of purified and reconstituted proteins or expressed in oocytes. With aims 1 and 2 we expect to obtain general rules of conformational changes but also specific differences between Shaker and Ci-VSP. Aim 3: Conformational changes and kinetics of the eukaryotic sodium channels. In this period we aim at two general objectives. First, by using LRET with new fluorescent toxins and site- directed fluorescence we will measure conformational changes of each individual domain of Nav1.4 as a function of voltage with and without the ?1 subunit. Second, we will define the molecular basis of the fast kinetics of Na channels induced by the beta subunit, which is crucial for action potential generation, We will test the hypothesis that the ?1 subunit induces positive cooperativity. We will measure the distances between the ?1 subunit and ? subunit with LRET, and determine the number of ?1's per ? with LRET and single molecule fluorescence. This research is expected to impact our knowledge of how specific residues affect kinetics and steady-state properties that in many cases can be traced to mutations that cause epilepsy, arrhythmia, myotonias and sudden death.